Proteins are large and complex molecules. They are required to be in their native confirmation in order to remain biologically active and not be immunogenic. Proteins having a high pI value or distinct polarity may not show stability in solution at or around physiological pH conditions. Further, at high concentration, protein molecules in solution are susceptible to undergo aggregation or degradation or certain modifications with time during storage. Most common protein degradation pathways known from literature are protein aggregation, deamidation and oxidation [Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993)]. Degradation of protein during storage may take place due to chemical instability (i.e. any process which involves modification of the protein by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e. changes in the higher order structure of the protein). Chemical instability majorly can be result of deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result from denaturation, aggregation, precipitation or adsorption. In an aspect, the present invention discloses suitable formulation of antibody proteins. Antibodies are highly complex molecules and are the fastest growing class of biologics in the pharmaceutical industry due to their therapeutic effectiveness in humans. However, antibodies are subject to undergo aggregation or degradation or denaturation or chemical modifications resulting in the loss of biological activity during the manufacturing process and/or during storage with time. Such protein modifications can also make them immunogenic resulting in the generation of anti-drug antibodies by the patient which can reduce the drug availability during subsequent injections or worse induce an autoimmune reaction. Therefore, there is a need to have stable new formulations for antibody preparations which protect the molecules from aggregation or degradation or chemical modification during the manufacturing process and/or storage in liquid solution even at high protein concentration while preserving the active biological conformation of the antibody molecules.
Liquid pharmaceutical formulation is a primary choice for manufacturers to prepare a stable, safe and effective pharmaceutical preparation of antibody for therapy. Liquid pharmaceutical preparation is also considered to be easy-to-handle for the patients and by the patients. However, a long appreciated problem with liquid formulations of protein therapeutics is that of aggregation, where protein molecules physically stick together, for example, resulting in the formation of either soluble high molecular weight protein aggregates or insoluble protein aggregates, which may cause undesired immunological reactions in patients upon administration. Additionally, a major problem caused by the aggregate formation is that during the administration the formulation may block syringes or pumps rendering it unsafe to patients. Aggregation of protein can also significantly impact its potency, immunogenicity and stability. Another reason of degradation is that unfolding mediated adsorption at interfaces can often be an initiating step for irreversible aggregation in solution. In this respect, proteins tend to adsorb at liquid-solid, liquid-air, and liquid-liquid interfaces. Sufficient exposure of a protein's core at a hydrophobic surface can result in adsorption as a consequence of agitation, temperature or pH induced stresses. Further, proteins also are sensitive to, for example, pH, ionic strength, thermal stress, shear and interfacial stresses, all of which can lead to aggregation and result in instability. Another consequence of aggregation is particle formation, an important consideration in liquid and lyophilized protein pharmaceuticals.
WO 2004/016286 discloses a liquid formulation for stabilizing antibodies which treat TNF α mediated diseases, comprising the antibody, a buffer system, mannitol, polysorbates and tonicity agents. The formulation uses a citrate-phosphate buffer system. This formulation could be thawed/frozen at least 3 times without any detrimental effect on either the chemical and physicochemical properties or biological activity.
In order to realize the clinical potency of an antibody protein, there is a need for new and improved formulations comprising the antibody molecule(s) in its native conformation, which can be stored under a desired/suitable condition for long-term storage without formation of significant amount of aggregates or fragments or modified variants of the antibody protein, even at high protein concentration. The present invention addresses the above-identified need by providing novel stable formulations comprising an antibody molecule, preferably a monoclonal antibody, along with suitable excipients which makes the formulation stable and having sufficiently low viscosity at around physiological osmolality and which is therefore suitable for administration to mammals, particularly human subjects.
We herein disclose some such formulations which prevent the formation of aggregates during and after formulation while providing a suitable condition for long term storage.